Cleaning solution for automated analyzers

ABSTRACT

The invention relates to a novel cleaning solution for use particularly with automated analyzers used in clinical laboratories and a method of cleaning a surface with the cleaning solution. The solution eliminates problems of cross-contamination of samples due to reagent carryover which can be brought about by an analyzer&#39;s probe that dispenses more than one reagent. The solution is particularly suitable for resolving carryover problems in coagulation assays performed with automated systems. The cleaning solution includes a bile salt, an ionic surfactant, sodium ions, and water and has a pH in a range of about 1 to 4.

This application is a continuation-in-part of U.S. application Ser. No.08/141,441 filed on Oct. 21, 1993, and now U.S. Pat. No. 5,395,545,issued on Mar. 7, 1995.

DESCRIPTION OF THE INVENTION

This invention relates to a novel cleaning solution for use particularlywith automated analyzers used in clinical laboratories and a method ofcleaning a surface with the novel cleaning solution. This solutionremoves problems of cross contamination of samples due to reagentcarryover, brought about by the analyzer's probe that dispenses morethan one reagent. In particular, this solution resolves carryoverproblems in coagulation assays performed with automated systems.

BACKGROUND OF THE INVENTION

Thrombin, thromboplastin and phospholipids are all common ingredients inreagents used for coagulation assays performed on samples of serum andplasma. Thrombin and thromboplastin in particular, are very stickysubstances and are difficult to remove from a surface. Because of thisproperty, it is difficult to avoid cross contamination of a secondsample by the reagent used in one test that is still adhering to theprobe that is then used to deliver a different reagent to a secondsample. Cross contamination of a reagent for one assay into a reagentfor another assay or into a sample will adversely affect assay results.

This was not a problem when all coagulation assays were done manually,as separate pipettes were used with each reagent and with each sample. Apipette was discarded after each use, thereby eliminating crosscontamination problems.

Today, many coagulation assays are performed on analyzers. In mostanalyzers that have limited random access capabilities, crosscontamination problems are avoided by having dedicated fluidic pathwaysfor each reagent. By doing so, the same reagent is constantly dispensedby the same probe or pipette, generally in the same order for a largebatch of serum or plasma samples having the same test run. Therefore,the probe or pipette does not have to be cleaned, or cleaned well,between each dispensation of reagent, as the probe or pipette willalways be dispensing the same reagent.

However, the next generation of automated coagulation analyzers containsrandom access capabilities. This means that a limited number of probesattached to fluidic pathways will be dispensing a different reagent intoeach separate sample container, if the analyzer is so programmed.Automated analyzers that have random access capabilities are thereforesubject to cross contamination problems. For example, the presence ofthrombin from a fibrinogen assay and thromboplastin from a prothrombinassay on a probe results in a shortening of a samples clotting time inthe activated partial thromboplastin time assay. Thrombin,thromboplastin and fibrinogen are particularly difficult to remove froma surface because of their strong adhesion properties. Changes in assayresults would affect the diagnosis afforded a patient, thereby causingsevere ramifications to the patient's treatment.

Currently, there are some types of cleaners available that removecarryover. These are strong denaturing cleaners, such as sodiumdodecylsulfate, 10% bleach solutions or hydrogen peroxide solutions.Although they do remove carryover, these cleaners also denature thereagents at the same time, resulting in poor assay performance results.This occurs because the denaturing cleaners also remain on the probe andare carried back to the reagent vials or are mixed with the reagent asit enters the bore of the probe, prior to the dispensation of thereagent. Therefore, not only must each reagent be thoroughly cleanedfrom the probe, it must be rapidly cleaned in order for the probe to beable to dispense reagent into a large number of samples in a very shortamount of time, for example, 180 samples per hour.

A fully automated coagulation analyzer with random access capabilitiesto perform analyses related to hemostasis and thrombosis on serum andplasma samples uses common pathways for reagents, thereby necessitatinga substantially non-denaturing cleaning solution for the common reagentpathway, the probe.

Therefore, it is highly desirable in the art to have a solution forcleaning a reagent probe from residual coagulation assay reagents, inparticular, thrombin, thromboplastin and fibrin, in order to avoid anycontamination from the carryover of a reagent from one sample tube toanother.

SUMMARY OF THE INVENTION

This invention is a cleaning solution particularly suited to rapidlyremoving substantially all thromboplastin, thrombin, and phospholipidsfrom a surface. One surface that this solution cleans exceptionally wellis that of a probe used in automated analyzers, in particular those thatperform coagulation assays. The probe is cleaned of substantially all ofthromboplastin, thrombin, and fibrin that may have been present in thefirst sample or reagent carried by the probe, so much so that there isno detectable carryover to the next sample with which the probeinteracts.

The cleaning solution is an aqueous solution containing a bile salt, aninorganic salt and an anionic surfactant, having a pH of about 4 orless, preferably in the range of about 1 to 3. An organic acid may beused in the cleaning solution to maintain the pH in the desired range.

The invention also embodies a method for cleaning a surface, making itsubstantially free of thromboplastin, thrombin, and phospholipids bywashing the surface with an aqueous cleaning solution containing a bilesalt, an inorganic salt and an anionic surfactant.

DESCRIPTION OF PREFERRED EMBODIMENTS

We have invented a novel cleaning solution that removes stronglyadhering substances, such as thrombin, thromboplastin, and phospholipidsfrom surfaces, without leaving a detectable residue on the surface. Inparticular, this cleaning solution works exceptionally well on surfacessuch as reagent probes used in automated coagulation analyzers. Thissolution works rapidly and is easily rinsed from the surface, leaving nodetectable carryover of reagent or solution to be found in the nextreagent or sample dispensed from the same probe. This is particularlyimportant in automated systems, as the number of samples tested per hourcan be as much as 180.

The cleaning solution is an aqueous solution of a bile salt compatiblewith anionic surfactants, and sodium ions. It may also contain anorganic acid. This combination of components results in a highlyeffective cleaning solution primarily for use in coagulation-basedassays, to remove substantially all thrombin, phospholipid andthromboplastin reagents.

Bile salts compatible with anionic surfactants, such as taurocholic acidand taurodeoxycholic acid, are the first components of the solution.These salts have been used to solubilize and/or stabilize membraneproteins of cells, depending on concentration. The bile salt must beused in a concentration where the final solution remains clear, that is,without a precipitate. It has been found that the range of bile saltuseable is from approximately 0.1% w/v to about 2.0% w/v of the finalsolution. At less than 0.1% and more than 2.0% w/v, it has been foundthat taurocholic acid precipitates out of solution. The preferred rangeof bile salt in the final solution is from about 0.5% to about 1.0%. Themost preferred concentration is 0.5% of the final solution. Theseconcentrations have been found to effectively remove thromboplastin,thrombin, and phospholipids from reagent probes when used in the finalcleaning solution formulation.

It has been found that anionic ethoxylated phosphorylated surfactantsproduce the best response in this cleaning solution. Other types ofanionics are usable, such as sodium dioctyl sulfosuccinate. The bilesalt used must be soluble in the surfactant, and the surfactant mustremain stable in solution and not be carried over on the probe.Sulfonated surfactants were found to destabilize and affect final testanalysis results. Cationic and nonionic surfactants were also found tobe ineffective in the final solution formulation.

Anionic surfactants are surface active agents with a negative charge.They are sold by a number of companies under many well known brandnames. For example, Karawet™ SB, a blend of phosphorylated ethyoxylates,is sold by Rhone-Poulenc Surfactants and Specialties, Dalton, Ga., USA.Another anionic surfactant useful in this formulation includes a sodiumdioctyl sulfosuccinate, Texwet™ 1001, manufactured by Intex ProductsInc., Greenville, S.C., USA. A preferred anionic ethoxylatedphosphorylated surfactant is Chemfac™ PC-099, sold by Chemax, Inc.,Greenville, S.C., USA. The range of surfactant in the final formulationranges from about 0.2% to about 2.0% w/v. The preferred amount is about1.5% w/v.

The cleaning solution formulation may also comprise an organic acid inorder to maintain the solution at a pH at or below 4. In particular,these are carboxylic acids, such as formic acid and acetic acid. It isbelieved that the low pH may aid in the decoupling of proteinaceousmaterial from phospholipids. The preferred range of organic acid isabout 0.2% to about 5.0% w/v, with the most preferred amount being about1.0%w/v. We have found the cleaning solution to be most effective whenmaintained at an acid pH. As bile salts and surfactants used in thecomposition may be acidic, the quantity of these ingredients may beadjusted to maintain a pH in the preferred range. If necessary, the pHof the solution may be lowered using organic or inorganic acids, orraised using basic compounds. The goal is to maintain the pH at a valueless than about 4, preferably in the range of about 1-3, most preferablyat about 2.

Sodium ions are also integral to the formulation. One way of introducingthem into the formulation is through the use of sodium chloride, sodiumsulfate or sodium formate. Although other ions appear to be useable tosome degree, such as calcium, sodium ions are part of the optimumformulation. The preferred range of sodium chloride is about 0.5% toabout 5.0% w/v, with the most preferred amount being about 3.0% w/v.

The most preferred formulation of the cleaning solution is an aqueoussolution of formic acid, 1.0%; taurocholic acid, 0.5%; sodium chloride,3.0%; and Chemfac™ PC-099, 1.5%. All percentages are in weight/volume(gm/100 ml). This formulation removes thrombin, thrombo-plastin, andphospholipids from probes used in automated coagulation analyzers in arapid and thorough manner.

A less preferred formulation is formic acid, 0.5% w/v; taurocholic acid,0.5% w/v; sodium chloride, 3.0% w/v; and Chemfac™ PC-099, 0.75% w/v.

The preferred solution can be prepared in the following manner.

Using an appropriately sized container, add 0.8 liter of purified waterand begin mixing. Next, add in a range from approximately 0.2% w/v toabout 5.0% w/v of the organic acid, preferably 1.0% w/v of formic acid,to the mixing water and continue mixing until dissolved, approximately10 minutes. Slowly add the sodium ions as sodium chloride in a rangefrom about 0.5% w/v to about 3.0% w/v, most preferably 3.0% w/v ofsodium chloride, and mix for approximately 10 minutes or untildissolved. Slowly add to this solution the bile salts as taurocholicacid, in a range from approximately 0.1% w/v to about 2.0% w/v, mostpreferably 0.5% w/v of taurocholic acid, and mix for approximately 15minutes or until dissolved. Add an anionic surfactant to the solution ina range from approximately 0.2% w/v to about 2.0% w/v. A preferredsurfactant is Chemfac™ PC-099 at approximately 1.5% w/v. Mix for about10 minutes. Using purified water, q.s. to 1 liter and mix forapproximately 10 minutes. At ambient temperature, check the pH of thesolution and bring it to pH 1.7±0.3. At this point a dye may be added.The final solution should be filtered to produce a clear liquid.

The following examples are provided to describe but not limit theinvention.

EXAMPLE 1 Preparation of the Preferred Washing Solution

This example describes the production of 300 liters of the washsolution.

240 liters of purified water were added to a 300 liter glass containerand stirred. Three liters of formic acid were slowly added to the waterand mixed at approximately 300 rpm until dissolved. To the solutionbeing stirred was added 9 kg of sodium chloride. Mixing continued atapproximately 380 rpm until the sodium chloride dissolved. 1.5 kg oftaurocholic acid was added and stirring continued until it dissolved.4.5 kg Chemfac™ PC-099 was added to the container and mixing continuedfor approximately 10 minutes. Water was added to bring the volume to 300liters and mixing continued for another 10 minutes. The pH was kept near1.7. 3.0 grams of a dye, Violamine R, was added to the container, whilemixing continued at approximately 200 rpm for about 30 minutes. Thesolution was then filtered through a 0.2 micron filter prior to use.

EXAMPLE 2 Reagent Carryover Studies

Experiments were performed to determine the amount of carryover thatoccurs when a particular reagent, thromboplastin, is used. Thiscarryover occurs when the assay order, in an automated analyzer, theMDA™ (Organon Teknika Corp., Durham, N.C., USA), testing for hemostasisand coagulation values, is to first assay a sample for Prothrombin Time(PT) followed by an assay on a sample for an Activated PartialThromboplastin Time (APTT). If carryover does occur, clotting occursmore quickly in the APTT assays as the thromboplastin carried over fromthe PT assay reacts with the proteins in the sample.

An experimental automated analyzer was used to perform these assays.This analyzer has random access capabilities and the order of assays tobe run can be programmed. Because of this capability, each probe on theanalyzer can deliver or aspirate any number of samples or reagents intovarious test wells.

The assays were run in the following order on the automated analyzer:

    ______________________________________                                        PT         MDA Verify 1 (4 replicates)                                        APTT       MDA Verify 1 (4 replicates)                                        PT         MDA Verify 2 (4 replicates)                                        APTT       MDA Verify 2 (4 replicates)                                        PT         MDA Verify 3 (4 replicates)                                        APTT       MDA Verify 3 (4 replicates)                                        ______________________________________                                    

The reagents used were MDA™ Simplastin L, a liquid thromboplastin; MDA™Platelin LS; MDA™ Platelin L CaCl₂ ; water used as the Probe Cleaner;MDA Verify™ 1; MDA Verify™ 2; and MDA Verify™ 3. The MDA and Verifytrademarks are that of Organon Teknika Corporation, Durham, N.C., USA.MDA Verify™ 1, 2 and 3 are plasma controls readily available fromOrganon Teknika Corporation.

For the PT assay, an aliquot of MDA Verify™ 1 was aspirated from itscontainer by the first probe, Arm 1, and dispensed into a cuvette well.Each cuvette contained four wells. This was repeated three more times,in order to perform 4 replicates of the assay. After each sampling, Arm1 was rinsed with a priming solution. The cuvette was then moved down atrack to the next station, near Arm 4. Arm 4 aspirated an aliquot ofMDA™ Simplastin L and dispensed it to the first cuvette well, afterwhich Arm 4 was rinsed with water. This was repeated for each well ofthe cuvette. The cuvette was allowed to react for a short period of timeand was then moved by the track to the optics module, where eachreaction, a clot formation, was detected. The results of the detectionwere reported automatically.

As the PT assay was being run, the APTT assays began. An aliquot of MDAVerify™ 1 was aspirated from its container by the first probe, Arm 1,and dispensed into a cuvette well. Arm 1 was then rinsed with a primingsolution. This procedure was repeated three more times to supply a totalof four replicates of Verify™ 1 as sample tested. The cuvette was moveddown a track to the next station, near Arm 3, which then aspirated analiquot of MDA™ Platelin LS from the its container and dispensed it intothe first cuvette well, adding it to the sample. Arm 3 was then washedwith water. This step was repeated for each of the remaining threesamples. The cuvette was then moved to the next station, near Arm 4,which aspirated an aliquot of MDA Platelin™ L from its container anddispensed it into the first cuvette well. Arm 4 was then rinsed withwater. This step was repeated with each of the remaining three samples.The reaction was allowed to proceed and the cuvette was moved along thetrack to the optics module where the reaction was detected in each well.The results were reported automatically.

This procedure was repeated with MDA Verify™ 2 and 3 being run inquadruplicate, with the PT assay being performed first, followed by theAPTT assay. The results were obtained by calculating the % Differencefrom the mean of replicates 2-4 and replicate 1 on APTT assay using theformula: ##EQU1## A high % Difference indicates carryover ofthromboplastin.

The results of the assays are given in Table 1 below. The clotting timesare given in seconds. Std is one standard deviation limit, and % CV iscoefficient of variation. An acceptable range of results for these typesof assays is within 2 standard deviations.

                  TABLE 1                                                         ______________________________________                                        ASSAYS SAMPLE ID    REPLICATE CLOT TIME (sec.)                                ______________________________________                                        PT     MDA Verify 1 1         11.35                                                               3         11.22                                                               4         11.40                                                               Mean      11.32                                                               Std        0.07                                                               % CV       0.58                                           APTT   MDA Verify 1 1         30.87                                                               2         33.53                                                               3         33.52                                                               4         33.33                                                               Mean      32.81                                                               Std        1.12                                                               % CV       3.43                                                               % Diff     7.74                                           PT     MDA Verify 2 1         15.2                                                                2         15.2                                                                3         15.29                                                               4         15.29                                                               Mean      15.25                                                               Std        0.04                                                               % CV       0.30                                           APTT   MDA Verify 2 1         46.19                                                               2         56.79                                                               3         57.59                                                               4         57.38                                                               Mean      54.49                                                               Std        4.80                                                               % CV       8.81                                                               % Diff    19.32                                           PT     MDA Verify 3 1         21.51                                                               2         21.42                                                               3         21.54                                                               4         21.34                                                               Mean      21.45                                                               Std        0.08                                                               % CV       0.37                                           APTT   MDA Verify 3 1         58.63                                                               2         73.85                                                               3         77.61                                                               4         78.32                                                               Mean      72.10                                                               Std        7.96                                                               % CV      11.04                                                               % Diff    23.45                                           ______________________________________                                    

As can be seen from Table 1, the use of water as a probe cleanerresulted in faster, inaccurate clotting times in the APTT assays, aresult of the carryover of the thromboplastin used in the PT assaysaffecting the APTT assays. The standard deviations of the PT assayresults versus the APTT assay results are much lower and moreacceptable. In particular, the first sample of each APTT series reportssubstantially different results than do the remaining APTT assayresults.

EXAMPLE 3 Use of the Preferred Wash Solution

The wash solution as prepared in Example 1 was used in these experimentsas the MDA Probe Cleaner instead of the water used in Example 2. Allother reagents remained the same, and the procedure as described inExample 2 also remained the same.

The results of the PT and APTT assays are given in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        ASSAYS SAMPLE ID    REPLICATE CLOT TIME (sec.)                                ______________________________________                                        PT     MDA Verify 1 1         12.04                                                               2         12.14                                                               3         12.07                                                               4         12.08                                                               Mean      12.08                                                               Std       0.04                                                                % CV      -0.30                                           APTT   MDA Verify 1 1         33.23                                                               2         33.08                                                               3         33.17                                                               4         33.14                                                               Mean      33.15                                                               Std       0.05                                                                % CV      0.16                                                                % Diff    0.30                                            PT     MDA Verify 2 1         16.39                                                               2         16.52                                                               3         16.5                                                                4         16.59                                                               Mean      16.50                                                               Std       0.07                                                                % CV      0.43                                            APTT   MDA Verify 2 1         57.25                                                               2         58.25                                                               3         56.57                                                               4         58.56                                                               Mean      58.16                                                               Std       0.54                                                                % CV      0.93                                                                % Diff    2.07                                            PT     MDA Verify 3 1         22.22                                                               2         22.47                                                               3         22.14                                                               4         22.14                                                               Mean      22.24                                                               Std       0.14                                                                % CV      0.61                                            APTT   MDA Verify 3 1         77.33                                                               2         78.35                                                               3         78.01                                                               4         78.33                                                               Mean      78.01                                                               Std       0.41                                                                % CV      0.53                                                                % Diff    1.15                                            ______________________________________                                    

As shown in Table 2 above, no significant carryover of thromboplastin isseen. The wash solution removed detectable amounts of the thromboplastinfrom the probe, without itself affecting any assay results.

We claim:
 1. An aqueous cleaning solution comprising:a. bile salt; b.anionic surfactant; c. sodium ions; and d. water,wherein said solutionhas a pH in the range of about 1 to about 4 and removes substantiallyall of a reagent selected from the group consisting of thrombin,thromboplastin, and phospholipids from a surface.
 2. A solutionaccording to claim 1, wherein said bile salt is taurocholic acid in aconcentration range from about 0.1% w/v to about 2.0% w/v.
 3. A solutionaccording to claim 1, wherein said anionic surfactant is present in aconcentration range from about 0.2% w/v to about 2.0% w/v.
 4. A solutionaccording to claim 1, wherein said sodium ions are provided by sodiumchloride in a concentration range from about 0.5% w/v to about 5.0% w/v.5. A solution according to claim 4, wherein said concentration range ofsaid sodium chloride is from about 2.0% w/v to about 3.0% w/v.
 6. Asolution according to claim 1, wherein said solution removes all of saidreagent from the surface of a probe or pipette within a probe cleaningor washing time allowed on an automated coagulation analyzer.
 7. Amethod of cleaning a probe from residual thrombin, thromboplastin, orphospholipids adhering to or coating the probe, comprising washing aninterior of the probe with a solution according to claim 1, therebyremoving all traces of said thrombin, thromboplastin, or phospholipids.8. A method of cleaning a probe from residual thrombin, thromboplastin,or phospholipids adhering to or coating the probe, comprising insertingthe probe into a solution according to claim 1 and drawing up saidsolution inside the probe and expelling said solution from the probe,thereby removing all traces of said thrombin, thromboplastin, orphospholipids.